Oxazolidinone photoaffinity probes

ABSTRACT

Disclosed are novel compounds that are useful and effective as photoaffinity probes and methods of using oxazolidinone photoaffinity probes.

FIELD OF THE INVENTION

[0001] The present invention is directed, in part, to novel photolabileoxazolidinones and preparation thereof. The novel compounds can be usedas photoaffinity probes within sensitive bacteria including bothgram-positive and gram-negative bacteria and mammalian cells.

BACKGROUND OF THE INVENTION

[0002] A number of antibiotic compounds have been developed and shown tobe effective in inhibiting translation. The antibiotics neomycin,thiostrepton, and hygromycin appear to inhibit translocation andoccupation of the A site within a ribosome but have no effect onformation of the fMet-tRNA_(f) ^(Met)/ribosome translation complex noron the peptide-bond synthesis which occurs in the absence of elongationfactor P. Streptomycin, which causes misreading and also inhibits A-sitebinding, interacts with two sites on the 16S rRNA of the 30S subunit.Lincomycin inhibits peptidyltransferase and occupation of the A-site.Erythromycin inhibits peptidyltransferase and destabilizes thepeptidyl-tRNA/ribosome/mRNA complex.

[0003] A number of additional compounds have been recently developed andhave been shown to act as antimicrobial or antibacterial agents.International Publication WO 99/41244 discloses substituted aminophenylisoxazoline compounds useful as antimicrobial agents. U.S. Pat. No.5,910,504 describes hetero-aromatic ring substituted phenyloxazolidinoneantimicrobial agents. In addition, International Publication WO 00/10566discloses isoxazolinone antibacterial agents. An important step in thedevelopment of new antimicrobial or antibacterial agents, such as thosedisclosed above, is the elucidation of a mechanism of action. Thespecific site of interaction of non selective antibiotics/antitumoragents, such as sparsomycin, that inhibit protein translation by adifferent, less useful, and direct mechanism, has been described. Porseet al., Proc. Natl. Acad. Sci. USA, 1999, 96, 9003-9008. Previousstudies with chemical probes using isolated, cell-free systems havefailed to define the relative sites of interaction of these types ofantibiotic compounds of the oxazolidinone class. Matassova, et al., RNA,1999, 5, 939-946. This is, in part, because the previous methods wereincapable of defining the sites of the particular and specific mechanismof action of this important class of antibiotics. Probes that help toelucidate the mechanism of action of antimicrobial and/or antibacterialagents and methods of using the same are highly desired.

[0004] The present invention is directed, inter alia, to noveloxazolidinone compounds and use of oxazolidinone compounds asphotoaffmity probes. Compounds suspected of having antimicrobial and/orantibacterial activity can be used in intact cells and can be evaluatedfor a mechanism of action by using active and inactive enantiomers ofoxazolidinone compounds as competitors for crosslinking to componentswithin the cell. The compounds and methods of the present inventionallow one skilled in the art to elucidate the mechanism of action ofantibacterial and/or antimicrobial agents that are suspected ofinhibiting protein translation. These and other aspects of the inventionare described below.

SUMMARY OF THE INVENTION

[0005] The present invention is directed to novel compounds that areuseful and effective as photoaffmity probes useful for, inter alia,identification of the oxazolidinone binding site within gram-positiveand gram-negative bacteria. These compounds can also be used for aidingin the determination of oxazolidinone binding sites within mammaliancells.

[0006] The compounds of the present invention comprise Formula I shownbelow.

[0007] wherein X and Y are, independently, F, H or CH₃; R¹ is H or I; R²is H or OH; R³ is H or C₁-C₈ alkyl; L is a bond or —OCH₂C(═O); and Q is

[0008] wherein R⁴ is H, CH₃, CH₂CH₃ or cyclopropyl; and Z is O or S; ora pharmaceutically acceptable salt thereof.

[0009] Other compounds of the present invention comprise Formula IIshown below.

[0010] wherein X and Y are, independently, F, H or CH₃; R¹ is H or I; R²is H or OH; and Q is

[0011] wherein R⁴ is H, CH₃, CH₂CH₃ or cyclopropyl; and Z is O or S; ora pharmaceutically acceptable salt thereof.

[0012] Other compounds of the present invention comprise Formula IIIshown below.

[0013] wherein X and Y are, independently, F, H or CH₃; R⁵ is

[0014] wherein R⁶ is H, N₃, halogen, NH₂, OH, SH, C₁-C₄ alkylamino,C₁-C₄ dialkylamino, C₁-C₄ alkyl, nitrile, carboxamide, C₁-C₄ alkoxy,C₁-C₄ alkylthio, or C₁-C₄ alkoxycarbonyl; and P is

[0015] wherein Z is O or S; and R⁷ is

[0016] wherein R¹ is H or I; and R² is H or OH; or a pharmaceuticallyacceptable salt thereof.

[0017] In other embodiments of the invention, methods of using acompound comprising Formula IV as a photoaffinity probe are provided,wherein Formula IV is

[0018] wherein X and Y are, independently, F, H or CH₃; R⁸ is H or I; R⁹is H or OH; R¹⁰ is H or C₁-C₈ alkyl; L is a bond or —OCH₂C(═O); and Q is

[0019] wherein R¹¹ is H, CH₃, CH₂CH₃ or cyclopropyl; and Z is O or S; ora pharmaceutically acceptable salt thereof.

[0020] In other embodiments of the invention, methods of using acompound comprising Formula V as a photoaffinity probe are provided,wherein Formula V is

[0021] wherein X and Y are, independently, F, H or CH₃; R¹² is N₃ or

[0022] wherein R⁸ is H or I; R⁹ is H or OH; and Q is

[0023] wherein R¹¹ is H, CH₃, CH₂CH₃ or cyclopropyl; and Z is O or S; ora pharmaceutically acceptable salt thereof.

[0024] In other embodiments of the invention, methods of using acompound comprising Formula VI as a photoaffinity probe are provided,wherein Formula VI is

[0025] wherein X and Y are, independently, F, H or CH₃; R¹³ is

[0026] wherein R¹⁴ is H, N₃, halogen, NH₂, OH, SH, C₁-C₄ alkylamino,C₁-C₄ dialkylamino, C₁-C₄ alkl, nitrile, carboxamide, C₁-C₄ alkoxy,C₁-C₄ alkylthio, or C₁-C₄ alkoxycarbonyl; and P is

[0027] wherein: Z is O or S; and R¹⁵ is

[0028] wherein R⁸ is H or I; and R⁹ is H or OH; or a pharmaceuticallyacceptable salt thereof.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0029] Various definitions are made throughout this document. Most wordshave the meaning that would be attributed to those words by one skilledin the art. Words specifically defined either below or elsewhere in thisdocument have the meaning provided in the context of invention as awhole and as are typically understood by those skilled in the art.

[0030] As used herein, the term “cross-linking” or “binding” means thephysical interaction between the photoaffinity probe and at least onecomponent within a cell or from a cell or combinations thereof. Bindingincludes ionic, non-ionic, Hydrogen bonds, Van der Waals, hydrophobicinteractions, etc. The physical interaction, the binding, can be eitherdirect or indirect, indirect being through or because of another proteinor compound. Direct binding refers to interactions that do not takeplace through or because of another protein or compound but instead arewithout other substantial chemical intermediates.

[0031] As used herein, the term “component” within a cell means anyprotein, nucleic acid, lipid, etc. within a cell. Components include,but are not limited to, the contents of the cytoplasm, nucleus, cellmembrane, cell wall, and the like.

[0032] As used herein, the term “competitor compound” means anyidentifiable chemical or molecule, small molecule, peptide, protein,sugar, natural or synthetic, that is suspected (such as a test compound)to potentially interact with or compete with the photoaffinity probe forcross- linking to a component within a cell or from a cell.

[0033] As used herein, the term “contacting” means either direct orindirect, application of a photoaffinity compound or competitor compoundwithin a cell, on or to a cell, or to components from a cell. Thecompetitor compound or photoaffinity compound can be present within abuffer, salt, solution, etc.

[0034] As used herein, the term “oxazolidinone” means a compound of theclass known as oxazolidinones, including the compounds described in U.S.Ser. Nos. 07/438,759, 07/553,795, 08/006,596, 07/882,407, 07/786,107,07/831,213, 08/233,903, 08/119,279, 08/226,158, 08/155,988, 08/329,717,07/909,387, 08/339,979, 08/384,278, 08/875,800, 07/880,432, 08/610,031,08/332,822, 07/988,589, 08/003,778, 08/066,356, 08/438,705, 60/015,499,60/003,149, 09/138,205, 09/138,209, 08/696,313, 60/012,316, 08/803,469,60/003,838, 08/709,998, 60/008,554, 08/762,478, 60/007,371, 08/850,424,60/048,342, 09/080,751, 60/052,907, 60/064,746, 09/111,995, 60/064,738,60/065,376, 60/067,830, 60/089,498, 60/100,185, 09/081,164, 60/088,283,60/092,765, 07/244,988, 07/253,850; European Patents EP 0500686, EP0610265, EP 0673370; PCT Application Numbers PCT/US90/06220,PCT/US94/08904, PCT/US94/10582, PCT/US95/02972, PCT/US95/10992,PCT/US93/04850, PCT/US95/12751, PCT/US96/00718, PCT/US93/03570,PCT/US93/09589, PCT/US96/05202, PCT/US97/03458, PCT/US96/12766,PCT/US97/01970, PCT/US96/14135, PCT/US96/19149, PCT/US96/17120,PCT/US98/09889, PCT/US98/13437;and U.S. Pat. Nos. 5,700,799, 5,719,154,5,547,950, 5,523,403, 5,668,286, 5,652,238, 5,688,792, 5,247,090,5,231,188, 5,654,428, 5,654,435, 5,756,732, 5,164,510, 5,182,403,5,225,565, 5,618,949, 5,627,197, 5,534,636, 5,532,261, 5,776,937,5,529,998, 5,684,023, 5,627,181, 5,698,574, 5,220,011, 5,208,329,5,036,092, 4,965,268, 4,921,869, 4,948,801, 5,043,443, 5,130,316,5,254,577, 4,877,892, 4,791,207, 4,642,351, 4,665,171, 4,734,495,4,775,752, 4,870,169, 4,668,517, 4,340,606, 4,362,866, 4,193,918,4,000,293, 3,947,465, 4,007,168, 3,674,780, 3,686,170, 3,906,101,3,678,040, 3,177,114, 3,141,889, 3,149,119, 3,117,122, 5,719,154,5,254,577, 4,801,600, 4,705,799, 4,461,773, 4,243,801, 3,794,665,3,632,577, 3,598,830, 3,513,238, 3,598,812, 3,546,241, 3,318,878,3,322,712; the disclosures of each of which are incorporated herein byreference in their entirety. Preferred oxazolidinones include linezolidand eperezolid.

[0035] The present invention is directed to novel photoaffinity probescomprising Formula I, Formula II, or Formula III. The preferredconfiguration at C-5 is (S). It will be appreciated by those skilled inthe art that compounds of the present can have additional chiral centersand be isolated in optically active or racemic form. The presentinvention encompasses any racemic, optically-active (such asenantiomers, diastereomers), tautomeric, or stereoisomeric form, ormixture thereof, of a compound of the invention. The present inventionis also directed to compositions comprising photoaffinity probes whichcomprise Formula I, Formula II, or Formula III, or a mixture thereof.

[0036] Preferred compounds of this invention have one radioactiveelement which is either ³H (T₃), ³⁵S, or ¹²⁵I. It is understood,however, that the Formulas include all isotopic forms of the compoundsdepicted.

[0037] In some embodiments of the invention, compounds comprise FormulaI, shown below.

[0038] wherein X and Y are, independently, F, H or CH₃ in a variety ofsubstitution patterns. Preferred compounds have one fluorine and one H.R¹ is H or I. R² is H or OH. R³ is H or C₁-C₈ alkyl. L is a bond or—OCH₂C(═O). Q is

[0039] wherein R⁴ is H, CH₃, CH₂CH₃ or cyclopropyl. Z is O or S.Compounds comprising Formula I also include pharmaceutically acceptablesalts thereof.

[0040] Preferred compounds comprising Formula I have the followingsubstituents: X is F, Y is H, R³ is H, and R⁴ is CH₃. More preferably,compounds of Formula I include, but are not limited to,2-[4-[4-[(5S)-5-[(Acetylamino)methyl]-2-oxo-3-oxazolidinyl]-2-fluorophenyl]-1-piperazinyl]-2-oxoethyl-4-azido-2-hydroxy-5-iodo-¹²⁵I-benzoate,N-[[(5S)-3-[4-[4-(4-Azido-2-hydroxy-5-iodo-¹²⁵I-benzoyl)-1-piperazinyl]-3-fluorophenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide]-2-[4-[4-[(5S)-5-[(Acetylamino)methyl]-2-oxo-3-oxazolidinyl]-2-fluorophenyl]-1-piperazinyl]-2-oxoethyl4-azido-3-iodo-¹²⁵I-benzoate, andN-[[(5S)-3-[4-[4-(4-Azido-3-iodo-¹²⁵I-benzoyl)-1-piperazinyl]-3-fluorophenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide.

[0041] In other embodiments of the invention, compounds comprise FormulaII, shown below.

[0042] wherein X and Y are, independently, F, H or CH₃ in a variety ofsubstitution patterns. Preferred compounds have one fluorine and one H.R¹ is H or I. R² is H or OH. Q is

[0043] wherein R⁴ is H, CH₃, CH₂CH₃ or cyclopropyl. Z is O or S.Compounds comprising Formula II also include pharmaceutically acceptablesalts thereof.

[0044] Preferred compounds comprising Formula II have the followingsubstituents: X is F, Y is H, and R⁴ is CH₃. More preferably, compoundsof Formula II include, but are not limited to,N-[[(5S)-3-(4′-Azido-2-fluoro[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]-T₃-acetamide,N-[[(5S)-3-(4′-Azido-2-fluoro-3′-iodo[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]-T₃-acetamide,N-[[(5S)-3-(4′-Azido-2-fluoro-3′-iodo[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]ethane-³⁵S-thioamide,andN-[[(5S)-3-(4′-Azido-2-fluoro-3′-iodo-¹²⁵I-[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]acetamide.

[0045] In other embodiments of the invention, compounds comprise FormulaIII, shown below.

[0046] wherein X and Y are, independently, F, H or CH₃ in a variety ofsubstitution patterns. Preferred compounds have one fluorine and one H.R⁵ is

[0047] wherein R⁶ is H, N₃, halogen, NH₂, OH, SH, C₁-C₄ alkylamino,C₁-C₄ dialkylamino, C₁-C₄ alkyl, nitrile, carboxamide, C₁-C₄ alkoxy,C₁-C₄ alkylthio, or C₁-C₄ alkoxycarbonyl. P is

[0048] wherein Z is O or S. R⁷ is

[0049] wherein R¹ is H or I. R² is H or OH. Compounds comprising FormulaIII also include pharmaceutically acceptable salts thereof.

[0050] Preferred compounds comprising Formula III have the followingsubstituents: X is F, Y is H, and R⁶ is H. More preferably, compounds ofFormula III include, but are not limited to,(2E)-3-(4-azido-3-iodo-¹²⁵I-phenyl)-N-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-2-propenamide,4-azido-N-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-2-hydroxy-5-iodo-¹²⁵I-benzamide,andN-(4-azidophenyl)-N′-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-³⁵S-thiourea.

[0051] The present invention is also directed to methods of using acompound having Formula I, II, III, IV, V, or VI (shown above) as aphotoaffmity probe. Briefly, a cell, or component(s) thereof, iscontacted with a photoaffinity probe comprising Formula I, II, III, IV,V, or VI. Preferably, the photoaffinity probe is radiolabled. Thephotoaffinity probe is then exposed to light, preferably ultraviolet, inorder to activate the photoaffinity probe. Cross-linking of thephotoaffinity probe is determined by methods well known to those skilledin the art such as, for example, by detecting the radiolabel.Radiolabel, such as ³H, ¹²⁵I, or ³⁵S, for example, can be detected by avariety of autoradiography techniques well known to the skilled artisan.In other embodiments of the invention, the method further comprisescontacting the cell or component(s) thereof with a competitor compound.The ability of the competitor compound to interfere with cross-linkingof the photoaffmity probe indicates the bioactivity of the competitorcompound.

[0052] Cells of the present invention include, but are not limited to,gram positive bacterial pathogens, including, for example,Staphylococcus aureus; Staphylococcus epidermidis (A, B, C biotypes);Staphylococcus caseolyticus; Staphylococcus gallinarum; Staphylococcushaemolyticus; Staphylococcus hominis; Staphylococcus saprophyticus;Streptococcus agalactiae (group B); Streptococcus mutans/rattus;Streptococcus pneumoniae; Streptococcus pyogenes (group A);Streptococcus salivarius; Streptococcus sanguis; Streptococcus sobrinus;Actinomyces spps.; Arthrobacter histidinolovorans; Corynebacteriumdiptheriae; Clostridium difficle; Clostridium spps.; Enterococcuscasseliflavus; Enterococcus durans; Enterococcus faecalis;Enterococcusfaecium; Enterococcus gallinarum; Erysipelothrixrhusiopathiae; Fusobacterium spps.; Listeria monocytogenes; Prevotellaspps.; Propionibacterium acnes; and Porphyromonas gingivalis.

[0053] Cells also include, but are not limited to, gram negativebacterial pathogens, including, for example, Acinetobactercalcoaceticus; Acinetobacter haemolyticus; Aeromonas hydrophila;Bordetella pertussis; Bordetella parapertussis; Bordetellabronchiseptica; Bacteroides fragilis; Bartonella bacilliformis; Brucellaabortus; Brucella melitensis; Campylobacter fetus; Campylobacter jejuni;Chlamydia pneumoniae; Chlamydia psittaci; Chlamydia trachomatis;Citrobacter freundii; Coxiella burnetti; Edwardsiella tarda;Edwardsiella hoshinae; Enterobacter aerogenes, Enterobacter cloacae(groups A and B); Escherichia coli (to include all pathogenic subtypes)Ehrlicia spps.; Francisella tularensis; Haemophilusactinomycetemcomitans; Haemophilus ducreyi; Haemophilus haemolyticus;Haemophilus influenzae; Haemophilus parahaemolyticus; Haemophilusparainfluenzae; Hafnia alvei; Helicobacter pylori; Kingella kingae;Klebsiella oxytoca; Klebsiella pneumoniae; Legionella pneumophila;Legionella spps.; Morganella spps.; Moraxella cattarhalis; Neisseriagonorrhoeae; Neisseria meningitidis; Plesiomonas shigelloides; Proteusmirabilis; Proteus penneri; Providencia spps.; Pseudomonas aeruginosa;Pseudomonas species; Rickettsia prowazekii; Rickettsia rickettsii;Rickettsia tsutsugamushi; Rochalimaea spps.; Salmonella subgroup 1serotypes (to include S. paratyphi and S. typhi); Salmonella subgroups2, 3a, 3b, 4, and 5; Serratia marcesans; Serratia spps.; Shigellaboydii; Shigella flexneri; Shigella dysenteriae; Shigella sonnei;Yersinia enterocolitica; Yersinia pestis; Yersinia pseudotuberculosis;Vibrio cholerae; Vibrio vulnificus; and Vibrio parahaemolyticus.

[0054] Cells also include, but are not limited to, Mycobacterialspecies, including, for example, Mycobacterium tuberculosis;Mycobacterium avium; and other Mycobacterium spps.

[0055] Cells also include, but are not limited to, Mycoplasmas (orpleuropneumonia-like organisms), including, for example, Mycoplasmagenitalium; Mycoplasma pneumoniae; and other Mycoplasma spps.

[0056] Cells also include, but are not limited to, Treponemataceae(spiral organisms) including, for example, Borrelia burgdorferi; otherBorrelia species; Leptospira spps.; Treponema pallidum.

[0057] Methods for preparing the photoaffinity probes described inFormulas I, II, III, IV, V, and VI are depicted in the followingsynthesis schemes. It will be apparent to those skilled in the art thatthe described synthetic procedures are merely representative in natureand that alternative procedures are feasible and may be preferred insome cases.

[0058] Non-radioactive compounds of Formulas I and IV are prepared bythe methods described in Schemes A and B. As shown in Scheme A, couplingof a benzoic acid moiety (A₁) with an appropriate hydroxyacetylpiperazine fragment (A₂) leads to compounds A₃ of Formula I where L is—OCH₂C(═O). Coupling can be accomplished with1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride or anyother reagents familiar to ones skilled in the art. Appropriate benzoicacid fragments can be made by procedures known in the literature.(Dupuis, Can. J. Chem., 1987, 65, 2450-2453; Shu, J. Labelled Compoundsand Radiopharmaceuticals, 1996, 38, 227-237, each of which isincorporated herein by reference in its entirety). Appropriatehydroxyacetyl piperazine fragments can also be made by methods known inthe literature (Barbachyn, U.S. Pat. No. 5,547,950; Barbachyn, U.S. Pat.No. 5,990,136; and Synder, International Publication WO 00/10566-A1,each of which is incorporated herein by reference in its entirety).Methods for incorporation of ¹²⁵I into compounds A₃ are shown in SchemesC and D.

[0059] Non-radioactive compounds of Formulas I and IV where L is a bondare prepared by the synthetic sequence shown in Scheme B. An appropriatebenzoic acid fragment (A₁ of Scheme A) is coupled with an appropriatepiperazine (B₂) using 1,1-carbonyldiimidazole in tetrahydrofuran to givethe desired compound (B₃). Other coupling methods known to those skilledin the art are also possible. The piperazine fragment is made by methodsknown in the literature (Hutchinson, U.S. Pat. No. 5,700,799, which isincorporated herein by reference in its entirety; Barbachyn, U.S. Pat.No. 5,990,136; and Snyder, International Publication WO 00/10566-A1).Methods for incorporation of ¹²⁵I into compounds B₃ are shown in SchemesC and D.

[0060] Radioactive iodine is introduced into the compounds of Formulas Iand IV by the methods shown in Schemes C and D. Compounds C₂ of FormulaI (where R¹ is OH and R² is ¹²⁵I) are prepared by reaction of compoundsC₁ (prepared according to the methods of Schemes A and B) with Na¹²⁵Iand chloramine-T.

[0061] Alternatively, compounds D₃ of Formulas I and IV (where R¹ is Hand R² is ¹²⁵I) are prepared as shown in Scheme D. Reaction of compoundsD₁ (prepared by the methods shown in Schemes A and B) withhexamethylditin affords the stannanes D₂. Reaction of D₂ with Na¹²⁵I andchloramine-T leads to D₃.

[0062] Non-radioactive compounds of Formulas II and V are prepared bythe method shown in Scheme E. The appropriate biphenyl nitro fragment(E₁) is reduced in the presence of hydrogen gas and a palladium catalystto give the appropriate biphenyl aniline fragment (E₂). Other reductionmethods familiar to those skilled in the art may also be used.Conversion to the azido moiety (E₃) can be accomplished via displacementof the appropriate diazonium salt with sodium azide using conditionsfamiliar to those skilled in the art. The appropriate nitro fragments(E₁) can be prepared by methods known in the literature (Barbachyn, U.S.Pat. No. 5,654,435, which is incorporated herein by reference in itsentirety; Barbachyn U.S. Pat. No. 5,990,136; and Synder, InternationalPublication WO 00/10566-A1) or by other methods familiar to thoseskilled in the art. Introduction of radioactive elements into compoundsof Formulas II and V are depicted in Schemes F, G, and H.

[0063] Scheme F shows the procedure for incorporation of tritium intocompounds of Formulas II and V where Q is oxazolidinone, Z is O, and R⁴is CH₃. Reaction of F₁ (prepared according to Scheme E) with 6N HCl andmethanol affords the free amine F₂. Reaction of F₂ with tritiated sodiumacetate and a coupling reagent affords the tritiated acetamide F₃.Suitable coupling reagents includeO-benzotriazol-1-yl-N,N,N′,N′,tetramethyluronium hexafluorophosphate andO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate. Other acceptable coupling reagents are known bythose skilled in the art. Alternatively, tritiated acetic anhydride anda suitable base can be used in place of tritiated sodium acetate and acoupling reagent. Incorporation of tritium into compounds of Formulas IIand V where Q is isoxazoline is carried out in similar fashion.

[0064] Scheme G shows the method for incorporation of ³⁵S into compoundsof Formulas II and V where Q is oxazolidinone, Z is S, and R⁴ is CH₃.Reaction of F₂ (from Scheme F) with ethyl ³⁵S-dithioacetate affords the³⁵S-thioacetamide, G₂. Incorporation of ³⁵S into compounds of FormulasII and V where Q is isoxazoline is carried out in similar fashion.

[0065] Radioactive iodine can be introduced into compounds of FormulasII and V by the method shown in Scheme H. Reaction of H₁ (preparedaccording to the route shown in Scheme E) with hexamethylditin affordsthe organostannane H₂. Reaction of H₂ with Na¹²⁵I and chloramine-Taffords the radioiodinated compound H₃.

[0066] Compounds of Formula V where R¹² is N₃ are made according to theprocedures described in U.S. Pat. No. 5,910,504, Example 17, which isincorporated herein by reference in its entirety.

[0067] Scheme I illustrates a synthetic method for the preparation ofnon-radioactive compounds of Formulas III and VI where P isoxazolidinone, Z is O, and R⁷ is optionally substituted azidophenyl orazidocinnamoyl. Refluxing an appropriate acetamide fragment (I₁) inmethanolic hydrochloric acid affords the free amine I₂. The acetamidefragments (I₁) are prepared by methods known in the literature(Barbachyn, U.S. Pat. No. 5,565,571, which is incorporated herein byreference in its entirety; Barbachyn, U.S. Pat. No. 5,990,136; andSynder, International Publication WO 00/10566-A1). Coupling of I₂ withan appropriate benzoic acid fragment (I₃, n=0) or cinnamic acid fragment(I₃, n=1) leads to the amide I₄. Coupling can be accomplished with EDCor other reagents familiar to ones skilled in the art. Appropriatebenzoic acid fragments (I₃, n=0) are prepared by the same method used toprepare A₁ of Scheme A. Appropriate cinnamic acid fragments (I₃, n=1)can be prepared by coupling of an appropriate benzaldehyde withWittig-Horner reagents. Benzaldehyde fragments can be prepared byprocedures known in the literature (Shu, J. Labelled Compounds andRadiopharmaceuticals, 1996, 38, 227-237) or by other methods familiar tothose skilled in the art. Compounds of Formulas III and VI where P isisoxazoline or isoxazolinone are made by similar methods.

[0068] Introduction of ¹²⁵I into compounds of Formulas III and VI whereP is oxazolidinone, Z is O, and R⁷ is optionally substituted azidophenylor azidocinnamoyl is accomplished by the method shown in Scheme J.Reaction of I₄ (from Scheme I, where R¹ is H and R² is OH) with Na¹²⁵Iand chloramine-T affords the radioiodinated compound J₂. Introduction of¹²⁵I into compounds of Formulas III and VI where P is isoxazoline orisoxazolinone is carried out by similar methods.

[0069] Alternatively, introduction of ¹²⁵I into compounds of FormulasIII and VI where P is oxazolidinone, Z is O, and R⁷ is optionallysubstituted azidophenyl or azidocinnamoyl is carried out by the methodshown in Scheme K. Reaction of I₄ (from Scheme I, where R¹ is I and R²is H) with hexamethylditin affords the organostannane K₂. Reaction of K₂with Na¹²⁵I and chloramine-T affords the radioiodinated compound K₃.Radioiodination of compounds of Formulas III and VI where P isisoxazoline or isoxazolinone is carried out in similar fashion.

[0070] Scheme L illustrates a synthetic method for the preparation ofradioactive compounds of Formulas III and VI where P is oxazolidinone, Zis ³⁵S, and R⁷ is optionally substituted azidoaniline. An appropriate³⁵S-isothiocyanate L₂ is reacted with the appropriate aminomethylfragment I₂ (Scheme I) in refluxing THF to give the desired³⁵S-thiourea, (L₃). The required ³⁵S-isothiocyanate L₂ is prepared byreaction of an appropriate aniline with ³⁵S-thiophosgene. Introductionof ³⁵S into compounds of Formulas III and VI where P is isoxazoline orisoxazolinone is carried out in similar fashion.

[0071] The invention is further illustrated by way of the followingexamples which are intended to elucidate the invention. These examplesare not intended, nor are they to be construed, as limiting the scope ofthe disclosure.

EXAMPLES Example 1 Synthesis

[0072]2-[4-[4-[(5S)-5-[(Acetylamnino)methyl]-2-oxo-3-oxazohidinyl]-2-fluorophenyl]-1-piperazinyl]-2-oxoethyl4-azido-2-hydroxy-5-iodo-¹²⁵I-benzoate (Compound C₂ of Scheme C where Lis —CH₂C(═O), X is F, Y is H, Q is oxazolidinone, Z is O and R⁴ is CH₃)is prepared as follows.

[0073] Step 1. To a stirred solution of(S)—N-[[3-[3-fluoro-4[4-(hydroxyacetyl)-1-piperazinyl]phenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide(515.8 mg, 1.31 mmol) in dimethylformamide (10 ml) and pyridine (1 ml)is added 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride(509.9 mg, 2.66 mmol) followed by 4-azidosalicylic acid (Dupuis, Can. J.Chem., 1987, 65, 2450-2453) and a catalytic amount of4-dimethylaminopyridine. The reaction mixture is stirred at roomtemperature for 72 hours then concentrated. The residue is diluted withCH₂Cl₂ (100 ml) and is washed successively with H₂O (2×30 ml), 1 N HCl(2×30 ml), saturated NaHCO₃ (1×30 ml), dried (MgSO₄), filtered andconcentrated. The residue is dissolved in CH₃OH/CH₂Cl₂, absorbed ontosilica gel and is purified on a Biotage 40S column with a SIM using 2.5%CH₃OH in CH₂Cl₂ as the eluent to give 186.5 mg (0.33 mmol, 25%) of thebenzoate ester. mp 177-178° C. (dec). ¹H-NMR (DMSO)δ: 10.4, 8.24, 7.87,7.53, 7.17, 7.09, 6.76, 6.70, 5.17, 4.71, 4.09, 3.71, 3.60, 3.40, 3.02,2.96, 1.83.

[0074] Step 2. All reagents are prepared in 0.1 N NaPO₄ buffer, pH 7.4unless otherwise specified. Buffer (70 μl), chloramine-T (70 μl of a 1mM stock solution), and the azido phenol of Step 1 (10 μl of a 50 μMstock solution in DMSO) are added to a 1.5 ml glass reaction vial. Arubber septum cap is crimped onto the reaction vial and a solution of¹²⁵I₂ in sodium hydroxide (10 μl containing 1 mCi (Amersham #IMS 30) isadded. The reaction is gently vortexed in the dark for 2 hours at roomtemperature then quenched with 10% solution of sodium bisulfite (100μl). The quenched reaction is diluted with buffer (800 μl) andtransferred from the reaction vial with a 1 ml tuberculin syringe fittedwith an 18 gauge needle. The reaction volume (1 ml) is loaded onto apreconditioned C18 sep-pak cartridge (Millipore Corporation) and theunincorporated ¹²⁵I₂ is washed from the C18 resin with HPLC grade watercontaining 0.1% trifluoroacetic acid (20 ml). Product is eluted using of80% CH₃CN/0.1 TFA (3 ml). The typical yield of iodinated product isapproximately 30% of the total ¹²⁵I₂ added to the reaction.

Example 2 Synthesis

[0075]N-[[(5S)-3-[4-[4-(4-Azido-2-hydroxy-5-iodo-¹²¹I-benzoyl)-1-piperazinyl]-3-fluorophenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide(Compound C₂ of Scheme C where L is a bond, X is F, Y is H, Q isoxazolidinone, Z is O and R⁴ is CH₃) is prepared as follows.

[0076] Step 1. To a stirred suspension of(S)—N-[[3-[4-[3-fluoro-4-(1-piperazinyl)]phenyl]-2-oxo-5-oxazolidinyl]methyl]-acetamide(498.0 mg, 1.3 mmol) in CH₂Cl₂ (10 ml) is added diisopropylethylamine(0.70 ml, 4.0 mmol) followed by 4-azidosalicoyl chloride (342.6 mg, 1.7mmol) in CH₂Cl₂ (7 ml). The reaction mixture is stirred at roomtemperature for 18 hours and then is partitioned between CH₂Cl₂ (50 ml)and H₂O (10 ml). The phases are separated. The organic layer is washedwith H₂O (10 ml), dried (MgSO₄), filtered and concentrated. The residueis dissolved in CH₃OH/CH₂Cl₂, absorbed onto silica gel and is purifiedon a Biotage 40S column with a SIM using 3% CH₃OH in CH₂Cl₂ as theeluent to afford 412.3 mg (0.83 mmol, 62%) of the desired benzamide as atan solid. mp 188-189° C. (dec). ¹H-NMR (DMSO)δ: 10.2, 8.24, 7.51, 7.22,7.16, 7.07, 6.64, 6.58, 4.70, 4.07, 3.70, 3.36, 2.96, 1.83.

[0077] Step 2. Starting with the phenol prepared in Step 1, ¹²⁵I isintroduced according to the procedure described in Step 2 of example 1.

Example 3 Synthesis

[0078]2-[4-[4-[(5S)-5-[(Acetylamino)methyl]-2-oxo-3-oxazolidinyl]-2-fluorophenyl]-1-piperazinyl]-2-oxoethyl4-azido-3-iodo-¹²⁵I-benzoate (Compound D₃ of Scheme D where L is—CH₂C(═O), X is F, Y is H, Q is oxazolidinone, Z is O and R⁴ is CH₃)

[0079] Step 1. To a stirred solution of 4-azido-3-iodobenzoic acid(103.8 mg, 0.36 mmol, (Shu, J. of labelled Compounds andRadiopharmaceuticals, 1996, 38, 227-237)) in dry THF (2.0 ml) is added1,1-carbonyldiimidazole (58.2 mg, 0.36 mmol). The reaction mixture isstirred at room temperature for 1 hour, then(S)—N-[[3-[3-fluoro-4[4-(hydroxyacetyl)-1-piperazinyl]phenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide(141.9 mg, 0.36 mmol) is added followed by a catalytic amount of DMAP.The reaction mixture is heated at reflux for 72 hours. The reactionmixture was cooled to room temperature and poured into CH₂Cl₂ (30 ml)and washed successively with H₂O (15 ml), 1 N HCl (15 ml), saturatedaqueous NaHCO₃ (15 ml), brine (15 ml), dried (MgSO₄), filtered andconcentrated. The residue is purified on a Biotage 12M column using 2%CH₃OH in CH₂Cl₂ as the eluent to afford 119.6 mg (0.18 mmol, 50%) of thebenzoate ester. mp 137-139° C. ¹H-NMR (CDCl₃)δ: 8.52, 8.14, 7.49, 7.19,7.07, 6.95, 6.01, 5.00, 4.72, 4.02, 3.81, 3.75, 3.62, 3.05, 1.58.

[0080] Step 2. To a stirred solution of the iodobenzoate prepared inStep 1 (62.7 mg, 0.094 mmol) and hexamethylditin (46.3 mg, 0.14 mmol) indry TEF (3 ml) is added dichlorobis(triphenylphosphine)palladium (II)(2.0 mg, 0.003 mmol). The reaction mixture is degassed and is heated atreflux for 3 hours. The reaction mixture is cooled and filtered througha pad of celite. The filtrate is absorbed onto silica gel and purifiedon a Biotage 12M column with SIM using 2% CH₃OH in CH₂Cl₂ as the eluentto afford 28.6 mg (0.04 mmol, 43%) of the stannane. ¹H-NMR (DMSO)δ:8.25, 8.04, 8.00, 7.48, 7.42, 7.15, 7.10, 5.10, 4.73, 4.09, 3.71, 3.60,3.40, 3.02, 2.96, 1.83, 0.33.

[0081] Step 3. To a stirred solution of the stannane prepared in Step 2in dry acetonitrile is added a solution of 1M aqueous Na¹²⁵I followed bychloramine-T hydrate. After stirring at room temperature for 30 minutes,the reaction mixture is quenched with saturated aqueous Na₂S₂O₃ andpurified to give the radioiodinated material.

Example 4 Synthesis

[0082]N-[[(5S)-3-[4-[4-(4-Azido-3-iodo-¹²⁵I-benzoyl)-1-piperazinyl]-3-fluorophenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide(Compound D₃ of Scheme D where L is a bond, X is F, Y is H, Q isoxazolidinone, Z is O and R⁴ is CH₃)

[0083] Step 1. To a stirred solution of 4-azido-3-iodobenzoic acid(272.0 mg, 0.94 mmol) in dry THF (4 ml) is added 1,1-carbonyldiimidazole(152.6 mg, 0.94 mmol). The reaction mixture is stirred at roomtemperature for 1 hour, then(S)—N-[[3-[4-[3-fluoro-4-(1-piperazinyl)]phenyl]-2-oxo-5-oxazolidinyl]methyl]-acetamide(315.9 mg, 0.94 mmol) is added followed by DMF (2 ml). The reactionmixture is heated at reflux for 18 hours. The reaction mixture is cooledand poured into CH₂Cl₂ (40 ml) and successively washed with H₂O (20 ml),1 N HCl (20 ml), saturated aqueous NaHCO₃ (20 ml), brine (20 ml), dried(MgSO₄), filtered and concentrated. The residue is dissolved in CH₂Cl₂,absorbed onto silica gel and is purified on a Biotage 40S column withSIM using 2.5% CH₃OH in CH₂Cl₂ as the eluent to afford 376.7 mg (0.62mmol) of the benzamide as a yellow solid. ¹H-NMR (DMSO)δ: 8.24, 7.88,7.53, 7.47, 7.39, 7.19, 7.08, 4.71, 4.08, 3.70, 3.51, 3.40, 2.99, 1.83.

[0084] Step 2. To a stirred solution of the iodobenzamide prepared inStep 1 (82.4 mg, 0.13 mmol) and hexamethylditin (71.1 mg 0.22 mmol) indry THF (6 ml) is added tetrakis(triphenylphosphine)palladium(0). Thereaction mixture is degassed and heated at reflux for 12 hours. Thecooled reaction mixture is filtered through a plug of celite and thefiltrate is absorbed onto silica gel and purified on a Biotage 12Mcolumn with SIM using 2% CH₃OH in 49% CH₂Cl₂ and 49% EtOAC as the eluentto afford 28.2 mg (0.044 mmol, 34%) of the stannane. ¹H-NMR (DMSO) δ:8.24, 7.50, 7.43, 7.35, 7.18, 7.17, 4.71, 4.08, 4.01, 3.70, 3.40, 2.99,1.83, 0.32.

[0085] Step 3. To a stirred solution of the stannane prepared in Step 2in dry acetonitrile is added a solution of 1M aqueous Na¹²⁵I followed bychloramine-T hydrate. After stirring at room temperature for 30 minutes,the reaction mixture is quenched with saturated aqueous Na₂S₂O₃ andpurified to give the desired radioiodinated material.

Example 5 Synthesis

[0086]N-[[(5S)-3-(4′-Azido-2-fluoro[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]-T₃-acetamide(Compound F₃ of Scheme F where R¹ is H, R² is H, X is F, and Y is H)

[0087] Step 1. To a stirred solution of 4-iodonitrobenzene (6.86 g, 27.5mmol) in dry DMF (230 ml) is added bis(pinacolato)diboron (8.24 g, 32.4mmol) followed by potassium acetate (8.68 g, 88.5 mmol) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (624.6 mg,0.76 mmol). The reaction mixture is degassed and heated at 85° C. for 2hours. To the cooled dark reaction mixture is added(S)—N-[[3-(3-fluoro-4-iodophenyl)-2-oxo-5-oxazolidinyl]methyl]acetamide(5.8 g, 15.3 mmol) followed by 2 N aqueous Na₂CO₃ (143 ml) and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (312.0 mg,0.38 mmol). The reaction mixture is degassed and heated at 85° C. for 3hours. The cooled reaction mixture is partitioned between EtOAC (500 ml)and H₂O (300 ml). The phases are separated. The aqueous layer isextracted with EtOAC (300 ml). The organic layers are combined andsuccessively washed with H₂O (500 ml), brine (500 ml), dried (MgSO₄),filtered and concentrated. The residue is dissolved in CH₃OH/CH₂Cl₂,absorbed onto silica gel and is purified on a Biotage 40 M column (2lots) with SIM using 75% EtOAC in CH₂Cl₂ to 100% EtOAC as the eluent toafford 3.74 g (10.0 mmol, 65%) of the desired nitrobiphenyl compound.¹H-NMR (DMSO) δ: 8.30, 7.83, 7.68, 7.50, 4.78, 4.18, 3.80, 3.44, 1.84.

[0088] Step 2. A mixture of the nitrobiphenyl compound prepared in Step1 (3.74 g, 10.0 mmol), 10% palladium on carbon in THF (100 ml), CH₃OH(100 ml) and CH₂Cl₂ (100 ml) is hydrogenated under a balloon of hydrogenfor 18 hours. The reaction mixture is filtered through a pad of celiteand the filtrate is concentrated to afford 2.50 g (7.3 mmol, 73%) of thedesired aminobiphenyl. ¹H-NMR (DMSO)δ: 8.26, 7.45, 7.34, 7.23, 6.64,5.28, 4.73, 4.14, 3.76, 3.42, 1.84.

[0089] Step 3. To a stirred solution of the aminobiphenyl prepared inStep 2 (508.93 mg, 1.48 mmol) in CH₃OH (40 ml) and 1 M HCl (40 ml),cooled to 0° C. is added a 1.2 M aqueous NaNO₂ solution (1.48 ml, 1.78mmol). The reaction mixture is stirred at 0° C. for 90 minutes, thensulfamic acid (143.5 mg, 1.48 mmol) is added followed by sodium azide(115.4 mg, 1.78 mmol) in H₂O (1.5 ml). The reaction mixture is stirredat 0° C. for 45 minutes, then diluted with CH₂Cl₂ (200 ml). The phasesare separated. The aqueous phase is extracted with CH₂Cl₂ (75 ml). Thecombine organic phases are dried (MgSO₄), filtered and concentrated. Theresidue is dissolved in CH₃OH/CH₂Cl₂, absorbed onto silica gel and ispurified on a Biotage 40S column with SIM using 10% CH₃OH in CH₂Cl₂ asthe eluent to afford 262.9 mg (0.71 mmol, 48%) of the desiredazidobiphenyl as a pale yellow solid. ¹H-NMR (DMSO) δ: 8.27, 7.58, 7.42,7.24, 4.76, 4.16, 3.78, 3.43, 1.84.

[0090] Step 4. The azidobiphenyl prepared in Step 3 (102.4 mg, 0.27mmol) in 6 N HCl (2 ml) and CH₃OH (6 ml) is heated at reflux for 18hours. The CH₃OH is removed in vacuo and the solid precipitate isisolated by filtration and is washed successively with H₂O (10 ml),ether (2×15 ml) then dried to afford 82.1 mg (0.23 mmol, 82%) of thedesired amine hydrochloride. ¹H-NMR (DMSO) δ: 8.30, 7.62, 7.42, 7.25,4.98, 4.25, 3.91.

[0091] Step 5. To a stirring solution of 0.57 mg (6.94 μmol, 250 mCi) oftritiated acetic acid sodium salt (American Radiolabeled Chemicals, lotno ARC 990519) in 1 ml of dry DMF and 2.71 mg (21 μmol) ofdiisopropylethylamine at room temperature, is added 6.94 μmol of 0.45MO-Benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU) in dry DMF. The solution instantly turned pale yellow and isstirred at room temperature for 10 minutes. The activated [³H]aceticacid sodium salt was then added to a stirring solution of 2.73 mg (7.5μmol) of the amine hydrochloride prepared in Step 4 in 2 ml of dry DMF.The reaction is stirred at room temperature for 4.5 hours, then allsolvents are removed by vacuum distillation at room temperature. Thecrude reaction mixture is purified on a preparative TLC plate (AnaltechSilica gel GF, 500 micron, 20 cm×20 cm plate), eluted with 8% methanolin dichloromethane. The desired band is scrapped. The product is elutedfrom the silica gel with 20% methanol in dichloromethane and filtered.The filtrate is concentrated under vacuum, and the residue is dissolvedin 65.5 ml of methanol to afford 94.4 mCi of the desired tritiatedmaterial (1.44 mCi/ml methanol, specific activity 57.37 mCi/mg (57.37Ci/mmol), radiochemical purity 99.5% by HPLC).

Example 6 Synthesis

[0092]N-[[(5S)-3-(4′-Azido-2-fluoro-3′-iodo[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]-T₃-acetamide(Compound F₃ of Scheme F where R¹ is H, R² is I, X is F, and Y is H)

[0093] Step 1. To a stirred solution of the aniline prepared in Step 2of Example 5 (284.9 mg, 0.83 mmol) in acetic acid (3 ml) is added iodinemonochloride (134.5 mg, 0.83 mmol) in acetic acid (0.25 ml). Thereaction mixture is stirred at room temperature for 1.5 hours. Thereaction mixture is partitioned between EtOAc and aqueous Na₂S₂O₃. Thephases are separated. The aqueous phase is extracted with EtOAc (20 ml).The combined organic phases were dried (MgSO₄), filtered andconcentrated. The residue is dissolved in CH₃OH, absorbed onto silicagel and is purified on a Biotage 40S column with SIM using 10-25%acetone in CH₂Cl₂ as the eluent to afford 48.4 mg (0.10 mmol, 12%) ofthe desired iodoaniline as a yellow oil. ¹H-NMR (CH₃OD) δ: 7.73, 7.52,7.29, 6.85, 4.81, 4.10, 3.79, 3.53, 3.32, 1.98.

[0094] Step 2. To a stirred solution of the iodoaniline prepared in Step1 (47.2 mg, 0.10 mmol) in CH₃OH (2 ml) and 1N HCl (2 ml) cooled to 0°C., is added a solution of NaNO₂ (8.5 mg, 0.12 mmol) in H₂O (1 ml). Theyellow reaction mixture is stirred at 0° C. for 30 minutes, then asolution of NaN₃ (8.0 mg, 0.12 mmol) in H₂O (1 ml) is added. Thereaction mixture is stirred at 0° C. for 1 hour, during which time ayellow precipitate formed. The solid is isolated by filtration andwashed with H₂O and dried to afford 41.0 mg (0.083 mmol, 83%) of thedesired iodoazidobiphenyl as a yellow solid. mp 173-175° C. (dec).¹H-NMR (DMSO) δ: 8.27, 7,98, 7.60, 7.42, 4.76, 4.16, 3.78, 3.43, 1.84.

[0095] Step 3. A mixture of 129 mg (0.26 mmol) of the iodoazidobiphenylprepared in Step 2 (129.0 mg, 0.26 mmol), CH₃OH (6 ml) and 1 N HCl (2ml) are heated at reflux for 48 hours. The cooled reaction mixture isconcentrated to afford quantitative yield the desired aminehydrochloride as a tan solid. ¹H-NMR (CH₃OD) δ: 7.96, 7.63, 7.46, 7.38,7.29, 5.04, 4.34, 3.93, 3.38, 1.30.

[0096] Step 4. To a solution of 5.1 mg (0.05 mmol, 25 mCi) of tritiatedacetic anhydride (Amersham Batch B77, isotope #00-0316) is added 2 NPCl₃ in CH₂Cl₂ (25 μl). The reaction mixture is left at room temperaturefor 5 hours with occasional mixing. To this mixture is added a solutionof the amine hydrochloride prepared in Step 3 (47.7 mg, 0.104 mmol) inpyridine (0.25 ml) followed by DMAP (4.6 mg). After 30 minutes, thereaction mixture is partitioned between H₂O and CH₂Cl₂. The phases areseparated. The aqueous phase is extracted exhaustively with CH₂Cl₂ andthen concentrated. The residue is purified on silica gel (4 g) using 20%acetone in toluene as the eluent to afford 38.2 mg (0.077 mmol, 74%) ofdesired tritiated acetamide.

Example 7 Synthesis

[0097]N-[[(5S)-3-(4′-Azido-2-fluoro-3′-iodo[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]ethane-³⁵S-thioamide(Compound G₂ of Scheme G where R¹ is H, R² is I, X is F, and Y is H)

[0098] Step 1. Methylmagnesium chloride in tetrahydrofuiran (THF) istreated with ³⁵S labeled carbon disulfide at 40° C., followed bytreatment with ethyl iodide. The reaction is stirred at 60° C. for 1.5hours. After workup with water and ethyl ether, the desired ethyl³⁵S-dithioacetate is obtained.

[0099] Step 2. The amine hydrochloride salt prepared in Step 3 ofExample 6 and the ethyl [³⁵S]dithioacetate prepared in Step 1 arestirred in methylene chloride, methanol, and triethylamine to give thedesired ³⁵S labeled thioamide.

Example 8 Synthesis

[0100]N-[[(5S)-3-(4′-Azido-2-fluoro-3′-iodo-¹²⁵I-[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]acetamide(Compound H₃ of Scheme H where X is F, Y is H, Q is oxazolidinone, Z isO, and R⁴ is CH₃)

[0101] Step 1. To a stirred solution of the iodobiphenyl prepared inStep 2 of Example 6 (56.2 mg, 0.11 mmol) and hexamethylditin (73.9 mg,0.22 mmol) in toluene (5 ml) is added palladium (II) acetate (2.6 mg,0.011 mmol) followed by triphenylphosphine (6.5 mg, 0.022 mmol). Thereaction mixture is degassed and heated at 80° C. for 20 hours. Thecooled reaction mixture is concentrated to one half the volume, thenpurified on a Biotage 12S column using 10-20% acetone in CH₂Cl₂ as theeluent to afford 53.5 mg (0.10 mmol, 89%) of the desired stannane.¹H-NMR (CDCl₃) δ: 7.53, 7.43, 7.30, 7.21, 6.07, 4.83, 4.11, 3.83, 3.73,2.05, 0.35.

[0102] Step 2. To a stirred solution of the stannane prepared in Step 1in dry CH₃CN and pH 7 phosphate buffer is added chloramine-T followed bya solution of 1M aqueous Na ¹²⁵I. After 30 minutes, the reaction mixtureis quenched with saturated aqueous Na₂S₂O₃ and purified to give thetitle compound.

Example 9 Synthesis

[0103](2E)-3-(4-Azido-3-iodo-¹²⁵I-phenyl)-N-[[(5S)-3-[3-fluoro-4-(4-pyridinyl]-2oxo-5-oxazolidinyl]methyl]-2-propenamide(Compound I₄ of Scheme I where R⁵ is 4-pyridyl, X is F, Y is H, n is 1,R¹ is ¹²⁵I and R² is H)

[0104] Step 1. To a stirred solution of oxalyl chloride (0.10 mL, 1.2mmol) in CH₂Cl₂ (1.5 ml), cooled to −78° C., is added dry DMSO (0.14 ml,1.97 mmol). After 10 minutes, a solution of 4-azido-3-iodobenzyl alcohol(217.0 mg, 0.79 mmol (Shu, J. of Labeled Compounds andRadiopharmaceuticals, 1996, 38, 227-237)) in CH₂Cl₂ (2.5 ml) is added.After 15 minutes, triethylamine (0.33 ml, 2.37 mmol) is added and thereaction mixture is allowed to warm to room temperature. The reactionmixture is poured into CH₂Cl₂ (30 ml) and washed successively withH₂O(20 ml), brine (20 ml), dried (MgSO₄), filtered and concentrated. Theresidue is purified on a Biotage 12M column using 10% EtOAC in hexane toafford 195.5 mg (0.72 mmol, 91%) of the desired aldehyde. ¹H-NMR (CDCl₃)δ: 9.9, 8.31, 7.93, 7.28.

[0105] Step 2. To a stirred solution of the aldehyde prepared in Step 1(190.0 mg, 0.69 mmol) in dry THF (1 ml) is addedtriethylphosphonoacetate (0.15 ml, 0.76 mmol) followed by lithiumhydroxide monohydrate (32.1 mg, 0.76 mmol). The reaction mixture isstirred at room temperature for 48 hours. The reaction mixture is pouredinto CH₂Cl₂ (40 ml) and successively washed with H₂O (20 ml), brine (20ml), dried (MgSO₄), filtered and concentrated. The residue is dissolvedin CH₂Cl₂, absorbed onto silica gel and purified on a Biotage 40S columnwith a SIM using 5% EtOAC in hexane as the eluent to afford 165.1 mg(0.48 mmol, 70%) of the desired ester. mp 93-94° C. ¹H-NMR (CDCl₃) δ:7.97, 7.56, 7.16, 6.40, 4.29, 1.35.

[0106] Step 3. To a stirred solution of the ester prepared in Step 2(66.7 mg, 0.19 mmol) in CH₃OH (2 ml) is added 1 N LiOH (0.19 ml, 0.19mmol). The reaction mixture is heated at reflux for 12 hours. The cooledreaction mixture is concentrated and used immediately.

[0107] Step 4.(S)—N-[[3-[3-fluoro-4-(4-pyridyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide(1.40 g, 4.25 mmol) in CH₃OH (62 ml) and 6 N HCl (31 ml) is heated atreflux for 18 hours. The reaction mixture is concentrated to afford 1.48g of the amine bis-hydrochloride salt. ¹H-NMR (DMSO) δ: 8.98, 8.62,8.26, 7.75, 7.56, 7.35, 5.76, 5.07, 4.28, 4.30, 3.27.

[0108] Step 5. The amine bis-hydrochloride (from Step 4) (68.2 mg, 0.19mmol), the lithium carboxylate prepared in Step 3,1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (72.8 mg,0.38 mmol) and 1-hydroxybenzotriazole hydrate (30.8 mg, 0.23 mmol) aredissolved in pyridine (2 ml) and stirred at room temperature for 72hours. The reaction mixture is concentrated. The residue is dissolved inCH₂Cl₂ (40 ml) and washed with H₂O (20 ml), brine (20 ml), dried(MgSO₄), filtered and concentrated. The residue is dissolved inCH₃OH/CH₂Cl₂, absorbed onto silica gel and is purified on a Biotage 12Mcolumn with SIM using 2% CH₃OH (saturated with NH₃) in CH₂Cl₂ as theeluent to afford 56.2 mg (0.096 mmol, 51%) of the desired cinnamide.¹H-NMR (DMSO) δ: 8.66, 8.48, 8.02, 7.64, 7.49, 7.40, 7.35, 6.70, 4.86,4.22, 3.84, 3.60.

[0109] Step 6. To a stirred solution of the iodocinnamide prepared inStep 4 and hexamethylditin in dry THF is addedtetrakis(triphenylphosphine)palladium(0). The reaction mixture isdegassed and heated at reflux for 12 hours. The cooled reaction mixtureis filtered through a plug of celite and the filtrate is absorbed ontosilica gel and purified on a Biotage 12M column with SIM to afford thestannane.

[0110] Step 7. To a stirred solution of the stannane prepared in Step 5in dry acetonitrile is added a solution of 1M aqueous Na¹²⁵I followed bychloramine-T hydrate. After stirring at room temperature for 30 minutes,the reaction mixture is quenched with saturated aqueous Na₂S₂O₃ andpurified to give the desired radioiodinated material.

Example 10 Synthesis

[0111]4-Azido-N-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-2-hydroxy-5-iodo-¹²⁵I-benzamide(Compound J₂ of Scheme J where R⁵ is 4-pynidyl, X is F, and Y is H, andn is 0)

[0112] Step 1. To a stirred suspension of the amine bis-hydrochloridesalt prepared in Step 4 of Example 9 (172.4 mg, 0.48 mmol) in pyridine(4 ml) and CH₂Cl₂ (1 ml) is added 4-azidosalicylic acid (128.9 mg 0.72mmol) followed by added 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (184.0 mg, 0.96 mmol) and 1-hydroxybenzotriazole hydrate(77.8 mg, 0.58 mmol). The reaction mixture is stirred at roomtemperature for 72 hours then concentrated. The residue is dissolved inCH₃OH/CH₂Cl₂, absorbed onto silica gel and purified on a Biotage 40Scolumn with SIM using EtOAC as the eluent to afford 44.8 mg (0.10 mmol,21%) of the benzamide as a tan solid. mp 200-202° C. (dec). ¹H-NMR(DMSO) δ: 12.5, 9.1, 8.66, 7.92, 7.70, 7.67, 7.61, 7.50, 7.46, 6.70,6.60, 4.93, 4.25, 3.92, 3.70.

[0113] Step 2. Starting with the phenol prepared in Step 1, ¹²⁵I isintroduced according to the procedure described in Step 2 of Example 1.

Example 11 Synthesis

[0114]N-(4-Azidophenyl)-N′-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-³⁵S-thiourea(Compound L₃ of Scheme L where R⁵ is 4-pyridyl, X is F, Y is H, R¹ is Hand R² is H)

[0115] To a stirred solution of the amine bis-hydrochloride (from Step 4of Example 9) in dry THF is added Hunig's base followed by³⁵S-4-azidophenylisothiocyanate in THF. The reaction mixture is heatedat reflux for 1 hour. The cooled reaction mixture is cooled and purifiedto give the desired thiourea.

[0116] As those skilled in the art will appreciate, numerous changes andmodifications may be made to the preferred embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention. The entiredisclosure of each publication cited herein is hereby incorporated byreference.

What is claimed is:
 1. A compound comprising the formula

wherein: X and Y are, independently, F, H or CH₃; R¹ is H or I; R² is Hor OH; R³ is H or C₁-C₈ alkyl; L is a bond or —OCH₂C(═O); and Q is

wherein: R⁴ is H, CH₃, CH₂CH₃ or cyclopropyl; and Z is O or S; or apharmaceutically acceptable salt thereof.
 2. A compound of claim 1wherein X is F, Y is H, R³ is H, and R⁴ is CH₃.
 3. A compound of claim 1wherein said compound is2-[4-[4-[(5S)-5-[(Acetylamino)methyl]-2-oxo-3-oxazolidinyl]-2-fluorophenyl]-1-piperazinyl]-2-oxoethyl-4-azido-2-hydroxy-5-iodo-¹²⁵I-benzoate.4. A compound of claim 1 wherein said compound isN-[[(5S)-3-[4-[4-(4-Azido-2-hydroxy-5-iodo-¹²⁵I-benzoyl)-1-piperazinyl]-3-fluorophenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide.5. A compound of claim 1 wherein said compound is2-[4-[4-[(5S)-5-[(Acetylamino)methyl]-2-oxo-3-oxazolidinyl]-2-fluorophenyl]-1-piperazinyl]-2-oxoethyl4-azido-3-iodo-¹²⁵I-benzoate.
 6. A compound of claim 1 wherein saidcompound isN-[[(5S)-3-[4-[4-(4-Azido-3-iodo-¹²⁵I-benzoyl)-1-piperazinyl]-3-fluorophenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide.7. A compound comprising the formula

wherein: X and Y are, independently, F, H or CH₃; R¹ is H or I; R² is Hor OH; and Q is

wherein: R⁴ is H, CH₃, CH₂CH₃ or cyclopropyl; and Z is O or S; or apharmaceutically acceptable salt thereof.
 8. A compound of claim 7wherein X is F, Y is H, and R⁴ is CH₃.
 9. A compound of claim 7 whereinsaid compound isN-[[(5S)-3-(4′-Azido-2-fluoro[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]-T₃-acetamide.10. A compound of claim 7 wherein said compound isN-[[(5S)-3-(4′-Azido-2-fluoro-3′-iodo[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]-T₃-acetamide.11. A compound of claim 7 wherein said compound isN-[[(5S)-3-(4′-Azido-2-fluoro-3′-iodo[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]ethane-³⁵S-thioamide.12. A compound of claim 7 wherein said compound isN-[[(5S)-3-(4′-Azido-2-fluoro-3′-iodo-¹²⁵I-[1,1′-biphenyl]-4-yl)-2-oxo-5-oxazolidinyl]methyl]acetamide.13. A compound comprising the formula

wherein: X and Y are, independently, F, H or CH₃; R⁵ is

wherein: R⁶ is H, N₃, halogen, NH₂, OH, SH, C₁-C₄ alkylamino, C₁-C₄dialkylamino, C₁-C₄ alkyl, nitrile, carboxamide, C₁-C₄ alkoxy, C₁-C₄alkylthio, or C₁-C₄ alkoxycarbonyl; and P is

wherein: Z is O or S; and R⁷ is

wherein: R¹ is H or I; and R²is H or OH; or a pharmaceuticallyacceptable salt thereof.
 14. A compound of claim 13 wherein X is F, Y isH, and R⁶ is H.
 15. A compound of claim 13 wherein said compound is(2E)-3-(4-azido-3-iodo-¹²⁵I-phenyl)-N-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-2-propenamide.16. A compound of claim 13 wherein said compound is4-azido-N-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-2-hydroxy-5-iodo-¹²⁵I-benzamide.17. A compound of claim 13 wherein said compound isN-(4-azidophenyl)-N′-[[(5S)-3-[3-fluoro-4-(4-pyridinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]-³⁵S-thiourea.18. A method of using a compound comprising the formula

wherein: X and Y are, independently, F, H or CH₃; R⁸ is H or I; R⁹ is Hor OH; R¹⁰ is H or C₁-C₈ alkyl; L is a bond or —OCH₂C(═O); and Q is

wherein: R¹¹ is H, CH₃, CH₂CH₃ or cyclopropyl; and Z is O or S; or apharmaceutically acceptable salt thereof, as a photoaffinity probe. 19.The method of claim 18 comprising the steps: contacting a cell orcomponent thereof with said compound, wherein said compound isradiolabeled; exposing said radiolabeled compound to light; anddetecting said radiolabel.
 20. The method of claim 19 further comprisingcontacting said cell or components thereof ?with a competitor compound.21. A method of using a compound comprising the formula

wherein: X and Y are, independently, F, H or CH₃; R¹² is N₃ or

wherein: R⁸ is H or I; and R⁹ is H or OH; and Q is

wherein: R¹¹ is H, CH₃, CH₂CH₃ or cyclopropyl; and Z is O or S; or apharmaceutically acceptable salt thereof, as a photoaffinity probe. 22.The method of claim 21 comprising the steps: contacting a cell orcomponent thereof with said compound, wherein said compound isradiolabeled; exposing said radiolabeled compound to light; anddetecting said radiolabel.
 23. The method of claim 22 further comprisingcontacting said cell or components thereof with a competitor compound.24. A method of using a compound comprising the formula

wherein: X and Y are, independently, F, H or CH₃; R¹³ is

wherein: R¹⁴ is H, N₃, halogen, NH₂, OH, SH, C₁-C₄ alkylamino, C₁-C₄dialkylamino, C₁-C₄alkyl, nitrile, carboxamide, C₁-C₄ alkoxy, C₁-C₄alkylthio, or C₁-C₄ alkoxycarbonyl; and P is

wherein: Z is O or S; and R¹⁵ is

wherein: R⁸ is H or I; and R⁹ is H or OH; or a pharmaceuticallyacceptable salt thereof, as a photoaffinity probe.
 25. The method ofclaim 24 comprising the steps: contacting a cell or component thereofwith said compound, wherein said compound is radiolabeled; exposing saidradiolabeled compound to light; and detecting said radiolabel.
 26. Themethod of claim 25 further comprising contacting said cell or componentsthereof with a competitor compound.